Various methods for analyzing bacterial genes have been developed and used, including Sanger sequencing method, RAPD (random amplified polymorphic DNA) method, RFLP (restriction fragment length polymorphism) method, but these methods still entail a problem in that they are time-consuming and use complex procedures.
The detection of bacterial diseases in the aquaculture field relies on bacterial culture methods that are time-consuming. Such bacterial culture methods may pose problems associated with detection errors because of a lack of objectivity, and bacterial culture methods that use selective medium have disadvantages in that they can detect only single bacteria and cannot achieve detailed classification. In order to increase the accuracy of detection, a biological analysis method (API test) is used, but it uses a complex procedure and requires a long reaction time, and thus cannot achieve rapid detection. Particularly, since a database about bacteria associated with aquatic animal diseases is insufficient and the detection accuracy is low because of the insufficiency of database, this analysis method is not used in the actual aquatic field. When molecular detection products are developed, which overcome such problems and can achieve detection within a few hours, they make it possible to prevent damage caused by bacterial diseases in an early stage.
Many kinds of fish such as tilapia, yellowtail, Oncorhynchus mykiss, and flatfishes, which are aquacultured worldwide, are infected with Edwardsiella tarda, Streptococcus iniae, Streptococcus parauberis and Lactococcus garvieae, which cause Edwardsiellosis and Streptococcosis, and are perished. Particularly, Streptococcus iniae may infect humans to cause cellulitis, and 20 or more human cases infected with Streptococcus iniae in humans through fishes were reported in the USA, Canada, Hongkong, Taiwan and Singapore, and thus it is important to discriminate the infectivity and pathogenicity of these bacteria.
Accordingly, there is a need for a method which can easily analyze the genotype-specific pathogenicity of bacteria by selecting a genotype and a genetic marker of pathogenic bacteria detected in infected fish and using the genotype and the genetic marker. When markers as described above are developed, these markers make it possible to accurately detect and discriminate bacteria causing Streptococcosis and Edwardsiellosis, which frequently occur in aquacultured fish such as flatfishes. Furthermore, these markers make it possible to accurately identify the bacteria, by which individuals infected with the bacteria could be detected in an early stage and the abuse of antibiotics in fish could be prevented to thereby reduce the production cost of fish.
Under this technical background, the present inventors have made extensive efforts to develop a method for discriminating the species of bacteria causing Streptococcosis and Edwardsiellosis in fish and detecting individuals infected with the bacteria. As a result, the resent inventors have identified genetic markers for discrimination and/or detection of Edwardsiella tarda, Streptococcus iniae, Streptococcus parauberis and Lactococcus garvieae, which are bacteria causing fish diseases, and have found that when peptide nucleic acids and primer pairs, specific for the genetic markers, are used to obtain different fluorescence amplification and melting curves depending on bacterial species, the bacteria causing fish diseases can be discriminated in a simple, rapid and accurate manner, thereby completing the present invention.